The invention is in the field of ligand-receptor assays, including immunoassays, used for determining the presence or absence of an analyte in a biological fluid. More particularly, the apparatuses and methods of the invention relate to establishing the identity of a test subject and determining the presence or absence of at least one analyte in a biological fluid taken from the test subject using a single test device.
For many years, those skilled in the art of ligand-receptor assays have sought an effective, inexpensive, and reliable device and method for detecting the presence and/or absence of antigens, antibodies, and the like. The art is replete with such devices.
For many years, those skilled in the art have also sought devices and methods for assuring that the sample being tested actually came from a certain individual. Many schemes, some elaborate, some simple, have been established to verify that the test sample was in fact produced by a particular individual. Similarly, many schemes exist to thwart matching a particular test result with a particular person.
For example, drug testing is now a routine procedure in athletics, prison, and the work place. The protocols involve testing individual fluid samples such as urine or blood to determine the presence of certain antibodies in the fluid sample; a positive result may be an indication of drug use.
Previously such testing has been accomplished by a series of tests which may involve shifting of the fluid being tested to different containers and removal of the fluid from the person being tested to a distant place. Oftentimes, fluid misplacement and/or substitution opened questions as to the chain of custody of the tested fluid. Thus, a problem which has occurred during such testing is that test fluids may be obtained from persons other than the person to be tested or that test fluids become mixed, lost, or cannot be specifically identified with that person after the test is returned from the laboratory. Also, these assays typically take too long to obtain timely results.
Other shortcomings are associated with many of the devices and methods currently used for the determination of analytes in biological fluids. Frequently, a certain level of skill is required to use or handle such devices to obtain satisfactory results. In addition, most of these devices require one or more additional reagents, and frequently a wash solution, to apply to the device during the course of the test. In many of these devices, due at least in part to the limited sensitivity of the devices, relatively large amounts of oftentimes expensive reagents or of test sample are required to give accurate results.
As noted above, the principles which form the basis for this type of detection is a person""s immune system, i.e., the inherent capability of a mammal to respond to a foreign molecule, typically a macromolecule. Hereinafter, any molecule which is capable of eliciting such a response will be referred to as an antigen, i.e., an antibody generator. The proteins and protein fragments which are produced in response to the antigen will be referred to as antibodies or immunoglobulins.
The present invention overcomes problems inherent in known assay devices and systems through the use of a specifically designed assay device, preferably an immunoassay device which allows the determination of the presence or absence of an analyte in a sample, and preferably specifically identifies the person providing the sample.
The present invention is directed to a testing method and device and more specifically to a method and device for detecting the presence of an analyte, most preferably specific antigens or specific antibodies in a biological fluid. The present invention preferably also provides for positively identifying the individual tested.
The present invention provides an easily handled, disposable testing pad and a disposable test device suitable for detecting the presence or absence of an analyte and preferably for identifying the test subject. For example, a finger of the test subject may be coated with a labelled ligand and the finger may be pressed on a membrane substrate having a specific immobilized ligand bed to capture an analyte from a biological fluid while simultaneously providing an inkless (specific binding) visible fingerprint of the test subject so that positive identification of the fluid donor is irrefutably obtained. The fingerprint of the fluid donor obtained on the membrane may also include information about analytes, such as drugs, present in his body fluids at the time of performing the test. This information can be recorded or stored for positive identification when needed.
It is an object of the invention to assay a test sample for the presence or absence of an analyte while also identifying the subject being tested. Thus, problems of misidentification, chain of title, and delays may be eliminated.
It is an object of the present invention to provide a device for rapidly determining the presence or absence of an analyte taken from a test subject. It is also an object of the present invention to provide a device for rapidly determining the quantitative amount of an analyte in a sample taken from a test subject. Yet other objects of the present invention are to provide a device and a method of determining the presence or absence of a drug or drug metabolite in a test sample taken from a test subject and to provide a device and method determining whether a test subject is an acute or chronic drug abuser.
Another embodiment of the present invention, where identifying the test subject by a fingerprint is not critical, involves applying the sample of body fluid or a labelled ligand to the membrane with the pressure of a transfer means or applicator that is part of the test device. In contrast to similar known devices which apply liquid test samples and/or reagents dropwise to the surface of a reaction medium, the present invention, in which liquid test samples and/or reagents are applied and maintained under pressure on the surface of the reaction medium for a short duration, requires smaller amounts of test sample and reagents and demonstrates higher sensitivities than known devices.
A device according to the present invention which uses this principle is an enclosed assay test device. A preferred embodiment is one in which the assay test device includes a housing, a reaction medium having at least one reaction zone located in the housing, a medium containing a signal-producing or analyte-indicating agent located in the housing, and a medium for containing a test sample located in the housing. Both the medium containing a signal-producing agent and the medium for containing a test sample located in the housing are movable independently between a first position in spaced relationship to the reaction medium and a second position in substance-transferrable contact with the reaction medium. Preferably this is a self-contained test device.
A particular embodiment of this invention includes an assay test device employing a two-part housing. In one embodiment of the device, a reaction medium having at least one reaction zone is mounted in a first part of the housing. In the second part of the two-part housing is located a medium for containing a portion of a test sample. The medium for containing a portion of a test sample and the reaction medium are so arranged in the two parts of the housing that when the housing parts are brought together in a closed relationship, there is substance transferrable contact between the medium for containing a portion of a test sample and the reaction medium. In addition to at least one reaction zone, the reaction medium preferably has a control zone which is provided to identify the test subject by a fingerprint or like means if desired. Such a device may be designed as a self-contained assay device. As used herein, the term xe2x80x9cself-containedxe2x80x9d means that the device may be used without the addition of additional reagents or solutions, or only requiring the dropwise addition of water.
Another embodiment employs a somewhat similar arrangement but a medium containing a signal-producing agent is substituted for the medium for containing a portion of a test sample.
A preferred device according to the above xe2x80x9cpressurexe2x80x9d principle is similar to the aforementioned devices in that a two-part housing is also employed in which a reaction medium having at least one reaction zone is mounted in a first part of the two-part housing. However, like one of these embodiments, in the second part of the two-part housing, there is located a medium containing a signal-producing agent, such as a labelled material which will indicate the presence of an analyte. The device also includes a removable medium for containing a portion of a test sample which is located intermediate the first and second parts of the housing and in substance-transferable contact with the reaction medium when the first and second parts of the housing are in a closed relationship in a first closed position of the housing. The reaction medium and medium containing an analyte-indicating agent (or signal-producing agent) are so positioned and arranged within their separate housing parts that when the parts of the housing are in a closed relationship in a second position, the reaction medium and means for transferring the analyte-indicating agent are in substance transferable contact with one another.
In using this device, a test sample drawn from a test subject is placed on a removable medium for containing and transferring a portion of a test sample which is placed in the two-part housing. The housing is then closed to a first position such that the removable test sample containing medium is in transferable contact with and applies pressure to the reaction medium. The housing is then opened and the removable medium for containing and transferring a test sample is removed from the housing. The housing is closed a second time to a second closed position such that the means for transferring an analyte-indicating agent is in transferable contact with and applies pressure to the reaction medium.
In another embodiment of the invention, various segregated areas of the reaction medium include a specific member of an immunological pair (MIP) which bind to or capture a specific analyte, such as a drug or drug metabolite. Different segregated areas may contain MIPs that are specific for different designated analytes, thereby allowing determinations for more than one analyte to be carried out in a single assay.
The methods and devices according to the invention are particularly beneficial in that only a small amount of signal-producing agent is required, in contrast to the conventional flow through cassettes or devices; applying pressure while maintaining direct contact on the surface of the test device maintains a localized concentration of reagents, particularly the signal-producing agent, which appears to increase the speed and sensitivity of the reaction, e.g., test results are obtained in a matter of seconds versus a matter of minutes or longer; and, in some embodiments, labeling a secondary antibody (as opposed to labeling the primary antibody or the antigen) decreases the reaction time of the assay because the antigen""s shape is not altered by being bonded to a label.
1. LIGAND-RECEPTOR ASSAY: any technique involving the detection of the complex formed between a ligand and a substance which binds to the ligand. Preferably, one member of the complex is an analyte. The preferred ligand-receptor assay is an immunoassay. Ligand-receptor assays may be used to determine the presence, absence, quantity, and concentration of ligands in biological fluids. Ligand-receptor assays may be competitive or non-competitive, homogeneous or heterogeneous, direct or indirect, or a combined detection technique, e.g., binding an antibody to a small chemical moiety such as biotin or dinitrophenol (DNP).
It is preferred that during the assay process, substantially all of at least one predetermined ligand or ligand receptor remains in a predetermined position. Any technique for immobilizing a ligand or ligand receptor is included in the scope of the present invention. In a preferred embodiment, a ligand or ligand receptor is bound or immobilized on or in a solid phase. Typical immobilization mechanisms include, but are not limited to, covalent binding, noncovalent binding, chemical coupling, physical entrapment, and adsorption.
2. LIGAND: refers to the analyte itself, or a substance which can be used to infer the presence of an analyte in a test sample. A ligand-receptor refers to the substances for which there is a specific binding partner, e.g., the ligand. As used herein, ligand and ligand-receptors may be members of an immunological pair (MIP). Ligands and ligand-receptors may include haptens, hormones, antigens, antibodies (including anti-antibodies and antibody fragments such as Fab or Fc), DNA, RNA, oligonucleotides, nucleic acids, and complexes or metabolites of any of the above. The ligand or the ligand-receptor may be labeled or unlabeled.
3. ANALYTE: the substance to be assayed, and, depending on the specific assay used, may be a ligand or a ligand-receptor. Exemplary analytes include but are not limited to drugs, proteins, haptens, hormones, metabolites of the aforementioned, and other molecules, alone or in combination with a protein. A hapten is a small molecular weight material, such as some drugs or drug metabolites, which typically first attach to a protein in order to be antigenic or immunogenic. For example, some drugs may be found in the body in both free and bound forms, which in turn provides the ability to distinguish between chronic drug abusers and acute drug abusers. In the case of the former, in many instances an antibody is formed in the body against the bound form which can also be detected as an analyte by the present invention.
4. BIOLOGICAL FLUID: any body fluid which may be tested to determine the presence or absence of an analyte, including, but not limited to blood or a blood component, saliva, urine.
5. Signal-producing AGENT: any agent used in a ligand-receptor assay which can be used to produce a detectable signal. The preferred signal-producing agents provide an easily visible signal, and more preferably a color.